The objectives of this study are to utilize recently developed methods for the successful in vitro culture of rat trophoblast to establish predictable relationships between certain structural and functional events that occur in the cultured trophoblast cell. These events will be correlated with the development and differentiation of the trophoblast in vivo and in vitro. We will also determine if these processes are regulated or occur autonomously. Rat trophoblast, in utero, hatches and the trophoectoderm then attaches to the endometrium and insinuates itself between epithelial cells. By the process of invasion the cells then penetrate the decidualized stroma to establish a relationship with maternal vascular sinuses. Blastocysts cultured in vivo follow a similar pattern. This provides a very advantageous model for analyzing trophoblast development by studying these processes in defined conditions which can be experimentally altered. After hatching trophoectoderm of cultured blastocysts attach to co-cultures of uterine stroma cells. Initially they insinuate themselves before invading the stromal monolayer. Cells cultured without a substration will grow out on the plastic dish. We propose to characterize these cellular functions of cultured trophoblast by analyzing available developmental landmarks. Microtubule assembly and distribution (morphogenesis and outgrowth) will be correlated with evidence of invasiveness (plasminogen activator) and with indices of biochemical differentiation (steroidogenesis of progesterone, testosterone and estradiol; production of rat placental lactogen). Realization of the objectives of these studies of structural and functional correlates of trophoblast development are strengthened by the availability of a productive model (in vitro culture of rat trophoblast). Additional reinforcement derives from preliminary observations in cultured trophoblast of microtubule distribution, synthesis of testosterone and estradiol and production of rat placental lactogen. These are described here for the first time.